Composition for prevention and treatment of diabetic complication

ABSTRACT

The present invention relates to a composition for the prevention and treatment of diabetic complications, which contains an extract obtained by: crushing and drying any one selected from  Euphorbiae  radix, gingered  Magnolia  bark, parched  Puerariae  radix and  Glycyrrhizae  radix; extracting the dried herbal material with alcohol or aqueous alcohol solution: filtering the extract; and concentrating the filtrate under reduced pressure. Alternatively, the composition contains a mixture of extracts from  Euphorbiae  radix, gingered  Magnolia  bark, parched  Puerariae  radix and  Glycyrrhizae  radix, the amount of each of the herbal extracts being 5-85% by weight based on 100% by weight of the herbal extract mixture. Alternatively, the composition contains magnolol obtained by: extracting  Magnolia  bark with aqueous 80% ethanol solution at ambient temperature for 24 hours; systematically separating the extract in the order of normal-hexane, ethyl acetate and normal-butanol; selecting the normal-hexane layer and separating the selected layer into pre-fractions by normal silica gel chromatography; comparing the pre-fractions with the standard form magnolol on TLC to determine a magnolol-enriched fraction; and separating magnolol from the magnolol-enriched fraction by silica gel column chromatography.

TECHNICAL FIELD

The present invention relates to a composition for the prevention andtreatment of diabetic complications. More particularly, the presentinvention relates to a pharmaceutical composition and functional foodeffective for inhibiting the production of advanced glycationendproducts (ACEs) to prevent and treat diabetic complications, whichcontains an extract from each of Euphorbiae radix, gingered Magnoliabark, parched Puerariae radix and Glycyrrhizae radix, or a mixture ofextracts from these herbal materials, or magnolol isolated from Magnoliabark, thus inhibiting the production of advanced glycation endproducts(ACEs).

BACKGROUND ART

Diabetes is one of important adult diseases all over the world, and inKorea, it reaches a prevalence rate of 7-8% with rapid economic growthand becomes an important cause of death of people in the 60-70-year-oldage group. Diabetic complication, a cause of death by diabetes, occursby the damage of almost all organs in the body at 10-20 years after theonset of diabetes, and is expressed as diabetic retinopathy, diabeticcataract, diabetic nephropathy, diabetic neuropathy, etc. Particularly,chronic diabetic nephropathy is the most important cause of end-stagerenal disease and cannot be treated by other therapies than blooddialysis therapy and organ transplantation. This diabetic complicationcan progress even when diabetes is cured so as to recover the normalblood glucose concentration. This diabetic complication is known to becaused mainly by advanced glycation endproducts (AGEs) irreversiblyproduced as a result of the nonenzymatic glycation of protein due to thecontinuation of a high blood glucose condition.

Mechanisms of causing this diabetic complication are broadly describedas the nonenzymatic glycation of protein, polyol pathways, oxidativestress, etc.

The nonenzymatic glycation of protein is caused by the nonenzymaticcondensation of the amino acid groups (e.g., lysine residues) of proteinwith glucose, which produces advanced glycation endproducts (AGEs). Thenonenzymatic glycation of protein can consist of the following twosteps: (1) the amino acid groups (e.g., lysine residues) of protein, andthe aldehydes or ketones of glucose, are subjected to nucleophilicaddition reaction without the action of enzymes so as to form Schiffbases as early-stage products, and ketoamine adducts adjacent to theSchiff bases are condensed with each other to produce reversibleAmadori-type early glycation products; and (2) by the continuation of ahigh blood glucose condition, the reversible Amadori-type earlyglycation products are rearranged without degradation and cross-linkedwith proteins, thus producing advanced glycation endproducts.

Unlike the reversible Amadori-type early glycation products, theadvanced glycation endproducts are irreversible. Thus, even when bloodglucose is recovered to a normal level, the endproducts, once produced,are accumulated in tissues during a protein existing period withoutdegradation, resulting in an abnormal change in the structure andfunction of the tissues (Vinson, J. A. et al., 1996, J. NutritinalBiochemistry 7: 559-663; Smith, P. R. et al., 1992, Eur. J. Biochem.,210: 729-739).

For example, glycated albumin, one of advanced glycation endproductsproduced by the reaction of glucose with various proteins, acts as animportant factor of causing chronic diabetic nephropathy. The glycatedalbumin is introduced into glomerular cells more easily thannon-glycated normal albumin, and a high concentration of glucosestimulates glomerular cells to increase the synthesis of extracellularmatrices. The excessive introduction of glycated albumins and theincrease of extracellular matrices result in the fibrillation ofglomerules. By such mechanisms, the glomerules are continuously damaged,eventually making the use of an extreme treatment method, such as blooddialysis or organ transplantation, unavoidable. Also, it was reportedthat, due to chronic diabetes, collagens are accumulated on the arterialwall, and basement membrane proteins are accumulated on glomerules bybinding to advanced glycation endproducts (Brownlee, M., et al., 1986,Sciences, 232, 1629-1632).

As described above, the nonenzymatic protein glycation leads to theglycation of basement membranes, serum albumins, lens proteins, fibrins,collagens, etc., and the advanced glycation endproducts cause anabnormal change in the structure and function of the tissues, resultingin chronic diabetic complications, such as diabetic retinopathy,diabetic cataract, diabetic nephropathy, diabetic neuropathy, etc.

Moreover, it is known that the advanced glycation endproducts producedin the nonenzymatic protein glycation also play an important role inaging (Monnier et all., Proc. Natl. Acad. Sci. USA, 81: 583, 1984; Leeet al., Biochem. Biophys. Res. Comm., 123: 888, 1984; Diabetologia, 38:357-394).

As described above, the advanced glycation endproducts produced in thenonenzymatic protein glycation are main factors in the progression ofdiabetic complication and aging. Thus, to prevent the progression ofdiabetic complication and aging, the production of advanced glycationendproducts need to be inhibited.

Currently, the only synthetic drug as a protein glycosylation inhibitoris aminoguanidine, a nucleophilic hydrazine, which prevents Amadoriproducts from crosslinking with proteins, by binding to the Amadoriproducts, so as to inhibit the production of advanced glycationproducts, thus delaying or preventing the development of diabeticcomplication (Brownlee, M., et al., 1986, Sciences, 232, 1629-1632;Edelstein, D. et al., 1992, Diabetes, 41, 26-29). The aminoguanidine,which is the most promising synthetic drug candidate for the preventionand treatment of diabetic complication, was developed up to third-phaseclinical trials but has the problem of causing toxicity upon long-termadministration. Thus, the development of safer drugs is now needed.

Accordingly, due to limitations in disease-treating agents with theexisting synthetic compounds and the problems of side-effects andtoxicity in the application of such treating agents, the development ofdisease-treating agents based on medicinal herbal formulations are nowactively conducted.

Thus, during studies on medicinal herbal materials for the preventionand treatment of diabetic complication and aging, the present inventorshave found that Euphorbiae radix, gingered Magnolia bark, parchedPuerariae radix and Glycyrrhizae radix are effective for inhibiting theproduction of advanced glycation endproducts, and particularly, amixture consisting of these herbal materials at an amount of each herbalmaterial of 5-85% by weight based on the total weight of the herbalmaterials taken as 100% by weight, and magnolol isolated from Magnoliabark, have an excellent effect on the inhibition of production ofadvanced glycation endproducts and thus, are useful for not only theprevention and treatment of diabetic complication but also theprevention and delay pf aging. On the basis of these findings, thepresent invention has been completed.

DISCLOSURE OF INVENTION Technical Problem

It is therefore an object of the present invention to provide acomposition for the prevention and treatment of diabetic complication,which contains, as an active ingredient, an extract from any oneselected from Euphorbiae Radix, gingered Magnolia bark, parchedPuerariae radix and Glycyrrhizae radix.

Another object of the present invention is to provide a composition forthe prevention and treatment of diabetic complication, which contains,as an active ingredient, a mixture of extracts from Euphorbiae radix,gingered Magnolia bark, parched Puerariae radix and Glycyrrhizae radix.

Still another object of the present invention is to provide acomposition for the prevention and treatment of diabetic complication,which contains, as an active ingredient, an extract from a mixture ofEuphorbiae Radix, gingered Magnolia bark, parched Puerariae radix andGlycyrrhizae radix.

Still another object of the present invention is to provide acomposition for the prevention and treatment of diabetic complication,which contains, as an active ingredient, magnolol isolated from Magnoliabark, or a pharmaceutically acceptable salt thereof.

Still another object of the present invention is to provide apharmaceutical composition for the prevention and treatment of diabeticcomplication, which contains, as active ingredients, said single herbalextract, said herbal extract mixture, or said isolated magnolol.

Still another object of the present invention is to provide a functionalfood for the prevention and treatment of diabetic complication, whichcontains, as active ingredients, said single herbal extract, said herbalextract mixture, or said isolated magnolol.

Still another object of the present invention is to provide apharmaceutical composition for the prevention and delay of aging, whichcontains, as active ingredients, said each single herbal extract, saidherbal extract mixture, or said isolated magnolol.

Still another object of the present invention is to provide a functionalfood for the prevention and delay of aging, which contains, as activeingredients, said each extract, said medicinal herbal mixture, or saidmagnolol.

TECHNICAL SOLUTION

To achieve the above objects, in one embodiment, the present inventionprovides a composition which contains an extract obtained by: crushingand drying any one selected from Euphorbiae radix, gingered Magnoliabark, parched Puerariae radix and Glycyrrhizae radix; extracting thedried herbal material with alcohol or aqueous herbal solution; filteringthe extract; and concentrating the filtrate under reduced pressure.

In another embodiment, the present invention provides a compositionwhich contains an herbal extract mixture obtained by: crushing anddrying each of Euphorbiae Radix, gingered Magnolia bark, parchedPuerariae radix and Glycyrrhizae radix; mixing the crushed herbalmaterials together, each of the herbal materials being used at an amountof 5-85% by weight based on the total weight of the herbal materialstaken as 100% by weight; extracting the herbal mixture with alcohol oraqueous alcohol solution; filtering the extract; and concentrating thefiltrate under reduced pressure.

In still another embodiment, the present invention provides acomposition containing extracts from Euphorbiae Radix, gingered Magnoliabark, parched Puerariae radix and Glycyrrhizae radix, in which theamount of each of the extracts is 5-85% by weight based on 100% byweight of the mixture of the extracts.

In yet another embodiment, the present invention provides a compositionwhich contains magnolol obtained by: extracting Magnolia bark withaqueous 80% ethanol solution at ambient temperature for 24 hours;systematically separating the extract in the order of normal-hexane,ethyl acetate and normal-butanol; separating the normal-hexane layerinto pre-fractions by normal silica gel chromatography; comparing thepre-fractions with the standard form magnolol on TLC to determine amagnolol-enriched fraction; and separating magnolol from themagnolol-enriched fraction by silica gel column chromatography.

Hereinafter, the medicinal herbal materials which are used in thepresent invention will be described.

Euphorbiae radix is the root of Elphorbia pekinensis, a perennial herbbelonging to Euphorbiaceae, and contains gallic acid, methylgallate,3-0-galloylshikimic acid, etc. (Kim, J. G., et al., Yakhak Hoeji, 1996,40, 170-176). It is bitter and pungent in taste, and cold in nature. Inthe spleen, lungs and stomach, it shows the effects of removing andexpelling retained water by hydrogogue and mitigating boils (Min-Kyo,Shin, Clinical Botany with Original Colors, Youngryumsa, 487, 1996).

Magnolia bark, which is the dried bark of Magnolia obovata, M.officinalis or M. officinalis var. biloba belonging to Magnoliaceae, hasthe effects of eliminating dampness and phlegm, and promoting thecirculation of “Gi”, and thus treating the following symptoms: drivingGi downward; retention of dampness and acupuncture if the diaphragm;numbness in skin and vomiting and diarrhea; retention of undigestedfood; abdominal distension and constipation; coughing caused by phlegm.(See The State Pharmacopoeia Commission of the People's Republic ofChina, Pharmacopoeia of the People's Republic of China, Chapter I, 204,Chemistry Industry Pressing, Beijing). Also, Magnolia bark containsessential oils, such as α, β, γ-eudesmol, magnolol, honokiol, alkaloid,saponin, etc. The known pharmacological effects of Magnolia bark includeanti-allergic effects (Shin, T. Y., et al., 2001, Arch. Pharm., Res.,24: 249-255), apoptotic effects (Park, H. J., et al., 2001, Arch.Pharm., Res., 24: 342-348), NO synthesis-inhibiting effects, TNF-αexpression-inhibiting effects (SOn, H. J., et al., 2000, Planata med.,66:467-471), antifungal effects (Bang, K. H., et al., 2000, Arch. Pharm,Res., 23: 46-49), mental health-promoting effects (Kuribara, H., et al,1999, J. Pharm. Pharmacol., 51: 97-103), and skin cancer-inhibitingeffects (Komoshima, T. et al., 1991, J. Nat. Prod., 54: 816-822).

Puerariae radix is the dried root of Pueraria thunbergiana (P. lobata),a perennial plant belonging to Fabaceae. It is sweet, pungent andordinary in taste. In the spleen and stomach, it shows the effects ofreleasing the exterior, eliminating bruised spots, producing the bodyfluid, and arresting diarrhea. The reported pharmacological actions ofPuerariae radix include fever alleviation, blood pressure lowering,memory enhancement, cerebral blood flow increase, coronary arterydilatation, heart function improvement, antiarrhythmic actions and thelike (Ho-Chul, Kim, Chinese medicinal pharmacology, JiipMoonDang, 92-94,2001).

Glycyrrhizae radix is obtained by drying the root and root stem ofGlycyrrhiza glabra, G. uralensis and others, which are perennial plantsbelonging to Fabaceae. It is sweet and ordinary in taste. In the spleen,stomach, heart and lungs, it shows the effects of strengthening thespleen and stomach, augmenting “Gi”, clearing heat, removing toxicity,moistening the lungs, alleviating and stopping a pain, and regulating“Gi”. It mainly contains glycyrrhizin (triterpen saponin), flavonoidcompounds, such as liquiritin, and the like. The pharmacological effectsof Glycyrrhizae radix include effects similar to those of adrenal cortexhormones, gastric ulceration inhibition, smooth muscle relaxation, liverfunction protection, anti-inflammation, anti-allergy, and anti-viruseffects (Ho-Chul, Kim, Chinese medicinal pharmacology, JiipMoonDang,92-94, 2001).

BRIEF DESCRIPTION OF DRAWINGS

FIG. 1 shows graphically the effect of the inventive herbal extractmixture on the inhibition of production of advanced glycationendproducts.

FIG. 2 shows graphically the effect of the inventive Euphorbiae radixextract on the inhibition of production of advanced glycationendproducts.

FIG. 3 shows graphically the effect of the inventive gingered Magnoliabark extract on the inhibition of production of advanced glycationendproducts.

FIG. 4 shows graphically the effect of the inventive parched PuerariaeRadix extract against the production of advanced glycation endproducts.

FIG. 5 shows graphically the inhibitory effect of the inventiveGlycyrrhizae radix extract on the inhibition of production of advancedglycation endproducts.

FIG. 6 shows graphically the effect of the inventive magnolol on theinhibition of production of advanced glycation endproducts.

FIG. 7 shows graphically the effect of aminoguanidine (cultured for 30days) on the inhibition of production of advanced glycation endproducts.

FIG. 8 shows graphically the effect of aminoguanidine (cultured for 90days) on the inhibition of production of advanced glycation endproducts.

FIG. 9 depicts photographs showing the eyeballs of test rats observed intest examples of the present invention.

FIG. 10 depicts photographs showing the eye lenses of test rats observedin test examples of the present invention.

FIG. 11 shows graphically the opacity of the eye lenses of test ratsmeasured in test examples of the present invention.

BEST MODE FOR INVENTION

Hereinafter, the present invention will be described in detail.

The inventive composition for the prevention and treatment of diabeticcomplication contains an extract obtained by the steps of: (1) dryingand crushing any one selected from Euphorbiae radix, gingered Magnoliabark, parched Puerariae radix and Glycyrrhizae radix, and thenextracting the crushed material with alcohol having 1-4 carbon atoms;and (2) filtering the extract obtained in the step (1), andconcentrating the filtrate under reduced pressure.

As the alcohol used in the step (1), although 10-90% alcohol having 1-4carbon atoms can be used, 80% ethanol is preferably used at an amount of5-10 times (w/v) the herbal medicinal material.

The gingered Magnolia bark used in the step (1) is prepared by treatingMagnolia bark in the following manner. To Magnolia bark in a containerpreheated to 50-100° C., ginger is added at an amount of about 3 partsby weight based on 100 parts by weight of Magnolia bark, and water isput in the container at an amount of 5-10 times (w/v) the herbalmaterial such that Magnolia bark is completely immersed in water. Then,the herbal material is heated while maintaining a temperature of 70-100°C. When water is almost evaporated, Magnolia bark is taken out from thecontainer. The gingered Magnolia bark thus obtained shows an increase ininhibitory effect against the production of advanced glycationendproducts as compared to Magnolia bark.

Parched Puerariae radix used in the step (1) is prepared by treatingPuerariae radix in the following manner. 100 g of Puerariae radix isparched at 120-130° C. for 45 minutes. As the surface of Puerariae radixbecomes yellow and shows brown spots, Puerariae radix is taken out andcooled. Parched Puerariae radix thus obtained shows an increase ininhibitory effect against the production of advanced glycationendproducts as compared to Puerariae radix.

Alternatively, the herbal extract mixture according to the presentinvention is obtained by mixing the extracts of the four herbalmaterials obtained in the steps (1) and (2), the amount of each of theherbal extracts being 5-85% by weight based on the total weight of theherbal extracts taken as 100% by weight.

Alternatively, the herbal extract mixture according to the presentinvention is obtained by: crushing and drying each of Euphorbiae radix,gingered Magnolia bark, parched Puerariae radix and Glycyrrhizae radix;mixing the crushed herbal materials, each of the herbal materials beingused at an amount of 5-85% based on the total weight of the herbalmaterials taken as 100% by weight; and extracting the herbal mixture inthe same manner as in the steps (1) and (2).

In in vitro tests, the inventive single herbal extract and mixed herbalextracts effectively inhibited the production of advanced glycationendproducts which cause diabetic complication. In animal tests, theywere excellent in blood glucose-lowering effects and showed an increasein body weight, a reduction in kidney hypertrophy, and remarkablereductions in BUN, triglyceride and creatinine levels, as compared to adiabetes-induced group. This suggests that they recover deterioratedkidney functions. In addition, they showed a remarkable reduction in theopacity of the eye lens as compared to the diabetes-induced group,indicating that they are effective for the delay or prevention ofcataract onset.

Also, magnolol according to the present invention is obtained byisolation from Magnolia bark, and represented by the formula (1) below.

Although magnolol, the index material of Magnolia bark, was known tohave an inhibitory effect against histamine release, its inhibitoryeffect against the production of advanced glycation endproducts wasfirst found in the present invention.

A method for isolating magnolol from Magnolia bark comprises the stepsof:

(1) extracting Magnolia bark with 80% ethanol aqueous solution atambient temperature for 24 hours; (2) systematically separating theextract in the order of n-hexane, ethyl acetate and n-butanol; (3)selecting the n-hexane layer and separating the selected n-hexane layerby normal silica gel (340 g) chromatography (using an n-hexane-ethylacetate mixture as a mobile phase) into pre-fractions; (4) comparing thepre-fractions with the standard form magnolol on TLC to determine amagnolol-enriched fraction; and (5) isolating magnolol from themagnolol-enriched fraction by normal silica gel chromatography. Magnololthus isolated is identified by analyses such as nuclear magneticresonance, mass spectrometry, IR, etc.

The isolated magnolol can be converted into a pharmaceuticallyacceptable salt according to any conventional method known in the art.For this purpose, pharmaceutically acceptable free acids which can beused in the present invention include inorganic acids and organic acids.Inorganic acids include hydrochloric acid, bromic acid, iodic acid,sulfuric acid and phosphoric acid, and organic acids include citricacid, acetic acid, lactic acid, tartaric acid, maleic acid, fumaricacid, formic acid, propionic acid, oxalic acid, trifluoroacetic acid,benzoic acid, gluconic acid, methanesulfonic acid, glycolic acid,4-toluenesulfonic acid, glutamic acid, and aspartic acid.

It was found that, in in vitro tests, the isolated magnolol inhibitedthe glycation of bovine serum albumin (BSA) and had the effect ofinhibiting the glycation of advanced glycation endproducts.

Accordingly, the inventive single herbal extract, mixed herbal extractand magnolol will be useful for the prevention and treatment of diabeticcomplications caused by the production of advanced glycationendproducts, for example, diabetic retinopathy, diabetic cataract,diabetic nephropathy, and diabetic neuropathy, etc.

Also, the inventive single herbal extract, mixed herbal extract, andmagnolol, inhibit the production of advanced glycation endproducts,resulting in reductions in the production of free radicals and theincidence of oxidative stresses. Thus, they will be useful for theprevention and delay of aging caused by oxidative stress.

The composition containing the inventive single herbal extract, mixedherbal extract or magnolol may additionally contain at least one activeingredient showing the same or similar function as these components.

In clinical administration, the inventive composition containing thesingle herbal extract or the herbal extract mixture may be administeredorally or parenterally, and used as a general drug formulation.

The composition containing the inventive single herbal extract or mixedherbal extract may be administered by various routes at effectiveamounts. The composition also contains a pharmaceutically acceptablecarrier. Any pharmaceutically acceptable carrier may be used if it isthe standard pharmaceutical carrier which can be used in knownformulations, such as sterilized solutions, tablets, coated tablets, andcapsules.

Generally, carriers include excipients, such as starch, milk, sugar,particular clays, gelatin, stearic acid, talc, vegetable oil, gum,glycol, and other known excipients. In addition, sweetening agents,pigment additives, and other components may also be included. Theinventive composition containing the inventive single herbal extract ormixed herbal extract as an active ingredient may be administered byvarious routes including but not limited to, oral, intravenous,intramuscular, transdermal routes, etc. In actual clinicaladministration, the inventive composition may be administered orally orparenterally in the form of various formulations, and it is formulatedwith generally used diluents or excipients, such as fillers, vehicles,binders, wettings, disintegrants, surfactants, etc.

Solid formulations for oral administration include tablets, pills,powders, granules, capsules and the like, and these solid formulationsare prepared with at least one excipient, such as starch, calciumcarbonate, sucrose, lactose, gelatin, etc. In addition to simpleexcipients, lubricants such as magnesium stearate and talc may also beused.

Liquid formulations for oral administration include suspensions,internal dosage forms, emulsions, syrups, etc., in which case simplediluents, such as water or liquid paraffin, and other variousexcipients, such as wetting agents, sweetening agents, aromatics, andpreservatives, may be used.

Furthermore, the inventive composition may be administered by parenteralroutes, such as subcutaneous, intravenous and intramuscular injections.In order to prepare formulations for parenteral administration, thesingle herbal extract or the mixed herbal extract is mixed with astabilizer or buffer in water to form a solution or suspension, which isthen formulated into unit-dose ampoules or vials.

The dose of the inventive single herbal extract or mixed herbal extractis suitably selected depending on the in vivo absorption, inactivationrate and excretion rate of the active ingredient, the age, sex andcondition of patients, and the severity of diseases to be treated. Itmay be administered 1-3 times daily. The effective dose of the inventivesingle herbal extract or mixed herbal extract contained in thecomposition is 500-2,000 mg/kg/day, and preferably 500-1,000 mg/kg/day.

The accurate dose, administration route and number of the inventiveformulation can be easily determined depending on the properties of theformulation, the bodyweight and condition of a subject, and theproperties of particular derivatives to be used.

In the present invention, the single herbal extract and the mixed herbalextract were tested for acute toxicity on test rats, and the resultsshowed that they had a minimum lethal dose (LD₅₀) of at least 6 g/kg,indicating no toxicity. This suggests that the inventive extract has avery high safety in the body. Accordingly, the inventive single herbalextract or mixed herbal extract may be administered safely to the body.

The inventive single herbal extract or mixed herbal extract may be usedfor the prevention and treatment of diabetic complications and for theprevention and delay of aging, alone or in combination with surgery,radiation therapy, hormonal therapy, chemical therapy, and/or methodsusing biological regulators.

The inventive single herbal extract or mixed herbal extract may be addedto foods for improving diseases caused by diabetic complication andaging. The inventive single herbal extract or mixed herbal extract maybe added to foods alone or in combination with other foods or foodadditives, and suitably used according to a conventional method.

The amount of addition of the active ingredient (herbal extract) can besuitably determined depending on the use purpose (prevention, healthpromotion or treatment). Generally, in the preparation of foods ordrinks, the inventive herbal extract mixture is added at an amount ofless than 15% by weight, and preferably 10% by weight, based on 100% ofraw materials. However, for the purpose of health and hygiene or in thecase of long-term intake for health control, the amount of addition ofthe inventive extract may be lower than the above-described amount.However, since the extract has no problem in view of safety, the activeingredient may also be used at a higher amount than the above-describedamount.

There are no special limitations on the kind of the foods. Examples offoods to which the extract may be added include dairy products, such asmeats, sausages, breads, chocolates, candies, snacks, confectioneries,pizzas, noodles, fried noodles, gums and ice creams, various soups,beverages, teas, drinks, alcoholic drinks and vitamin complexes, as wellas all foods which are functional in a common sense.

The inventive functional foods may additionally contain varioussweetening agents or natural carbohydrates as in conventional beverages.The natural carbohydrates include monosaccharides, such as glucose andfructose, disaccharides, such as maltose and sucrose, polysaccharides,such as dextrin and cycodextrin, and sugar alcohols, such as xylitol,sorbitol, and erythritol. Sweeteners include natural sweeteners such asthaumatin and stevia extracts, and synthetic sweeteners, such assaccharin and aspartame. The natural carbohydrates are used at an amountof about 0.01-0.04 g, and preferably about 0.02-0.03 g based on 100 mlof the inventive composition.

In addition, the inventive composition may contain various nutrients,vitamins, electrolytes, flavoring agents, colorants, pectic acid or itssalt, alginic acid or its salt, organic acids, protective colloidaltackifiers, pH adjusters, stabilizers, preservatives, glycerin, alcohol,carbonating agents used in carbonated drinks, etc. Also, the inventivecomposition may contain fruit flesh for the preparation of natural fruitjuices, fruit juice beverages and vegetable juices. These components maybe used alone or in combination. Although not critical, these additivesare used at an amount of 0.01-0.1 parts by weight based on 100 parts byweight of the inventive composition.

MODE FOR INVENTION

Hereinafter, the present invention will be described in further detailby the following examples and test examples. It is to be understood,however, that these examples are given for illustrative purpose only andare not construed to limit the scope of the present invention.

Example 1 Preparation of Medicinal Herbal Extract

Euphorbiae radix was powdered, and 100 g of the powder was taken andextracted with 1 liter of 80% ethanol aqueous solution (ethanol:distilled water=80: 20) at ambient temperature (20-30° C.) for 24 hours.The ethanol extract was filtered through filter paper, and then, theextraction and filtration steps repeated five times in the same manneras described above. All the extracts were collected and concentratedunder reduced pressure.

Meanwhile, Euphorbiae radix, 100 g of each of gingered Magnolia bark,parched Puerariae Radix and Glycyrrhizae radix was extracted with 1liter of 80% ethanol aqueous solution. Each of the extracts was filteredand concentrated under reduced pressure. As a result, obtained were 20 gof the Euphorbiae radix extract, 10 g of the gingered Magnolia barkextract, 20 g of the parched Puerariae radix extract, and 20 g of theGlycyrrhizae radix extract.

Example 2 Preparation of Herbal Extract Mixture

The extracts prepared in Example 1 were mixed together at an amount of10 g for each herbal extract, thus obtaining 40 g of an herbal extractmixture.

Example 3 Another Preparation of Herbal Extract Mixture

Euphorbiae radix, gingered Magnolia bark, parched Puerariae radix andGlycyrrhizae radix were powdered, and four kinds of the powers weremixed together at an amount of 100 g for each herbal material. Themixture was extracted with 1 liter of 80% ethanol aqueous solution(ethanol: distilled water=80:20) at ambient temperature (20-30° C.) for24 hours. The ethanol extract was filtered through filter paper, andthen, the extraction and filtration steps were repeated five times inthe same manner as described above. All the extracts were collected andconcentrated under reduced pressure.

Example 4 Isolation of Magnolol

2 kg of Magnolia bark was extracted with 10 liters of 80% ethyl alcoholaqueous solution at ambient temperature for 24 hours, followed byfiltration. The extraction and filtration steps were repeated threetimes, and all the resulting extracts were concentrated under reducedpressure and dried, thus obtaining 250 g (12.5% yield) of a dark-brownextract. This extract was systemically separated in the order ofn-hexane, ethyl acetate and n-butanol.

Of the separated layers, the n-hexane layer distributed with magnololwas selected and subjected to normal silica gel (340 g) chromatography,thus yielding pre-fractions. Here, the n-hexane layer was eluted with amobile phase of n-hexane:ethyl acetate at a gradient of 1:0 to 6:, thusyielding pre-fraction 1 to pre-fraction 6. The pre-fractions werecompared with the standard form magnolol and, as a result, thepre-fraction 4 was determined to be rich in magnolol. The pre-fractionwas subjected to silica gel chromatography, thus isolating 15 mg ofmagnolol. It was determined to be magnolol by the results of variousanalyses, including NMR, mass spectrometry and IR.

Test Example 1 Analysis of Effects of Inventive Single Herbal Extract,Mixed Herbal Extract and Magnolol on Inhibition of Production ofAdvanced Glycation Endproducts

In order to examine the effects of the inventive single herbal extract,mixed herbal extract and magnolol on the inhibition of production ofadvanced glycation endproducts, the following tests were carried out.

(1) Effect of Inventive Herbal Extract Mixture on Inhibition ofProduction of Advanced Glycation Endproducts

As a protein source, bovine serum albumin (hereinafter, referred to as“BSA”; Sigma, USA) was used. BSA was dissolved in 50 mM phosphate buffer(pH 7.4) to a concentration of 10 mg/ml.

As a sugar source, a mixture of 0.1 M fructose and 0.1 M glucose wasused.

This sugar mixture was added to the prepared BSA solution, for use intests.

The herbal extract mixture prepared in Example 2 was dissolved in 15%Tween80, and this solution was added to the BSA-sugar mixture andcultured at 37° C. for 30 days. At this time, 0.02% sodium azide wasused as an antibacterial agent.

As a control group, a culture consisting of BSA and the sugar mixturewas cultured, and as blanks to the control and test groups, thematerials for the test group and the materials for the control groupwere used after preparation without incubation.

In order to reduce errors to the lowest possible extent, 4 samples foreach group were used. Just before culturing, the samples were filledwith nitrogen gas (99.999% purity) in order to prevent them beingcontaminated. After 30 days, the content of advanced glycationendproducts in the culture media was analyzed. The advanced glycationendproducts are fluorescent, show a brown color, are crosslinkable, andhave ligands which can be recognized by cellular membrane receptors. Theamount of the advanced glycation endproducts with such properties wasmeasured with a spectrophotometer (excitation at 350 nm and release at450 nm) so as to determine the inhibition of production of the advancedglycation endproducts.

The inhibition (%) of production of advanced glycation endproducts wascalculated according to the following equation:Inhibition (%) of production=100−[(fluorescent intensity of testgroup−fluorescent intensity of blank to test group)/(fluorescentintensity of control group-fluorescent intensity of blank to controlgroup)]×100

(2) Effect of Extract from Each of Euphorbiae Radix, Gingered MagnoliaBark, Parched Puerariae Radix and Glycyrrhizae Radix, on Inhibition ofProduction of Advanced Glycation Endproducts

The part (1) of this Example was repeated except that extract from eachof Euphorbiae radix, gingered Magnolia bark, parched Puerariae radix andGlycyrrhizae radix was dissolved in 15% Tween80 and cultured for 90days.

(3) Effect of Magnolol on Inhibition of Production of Advanced GlycationEndproducts

Magnolol was dissolved in distilled water at concentrations of 3.33μg/ml, 6.66 μg/ml, 13.32 μg/ml and 26.6 μg/ml, and then cultured for 37°C. for 30 days in the same manner as in the above part (1).

aminoguanidine, a positive control group, was tested in the same manneras in the above part (1) except that aminoguanidine was dissolved indistilled water at selected concentrations and cultured for 30 days and90 days.

The test results are shown in FIGS. 1 to 8 and Table 1. TABLE 1Inhibition (%) of production of advanced glycation endproductsConcentration IC₅₀ (μg/ml) Inhibition (%) (μg/ml) Herbal extract mixture5 22.245 ± 0.698 18.12 (cultured for 30 days) 10 44.998 ± 1.396 2563.548 ± 2.234 50 93.238 ± 5.187 Euphorbiae radix extract 25 34.680 ±2.685 32.07 (cultured for 90 days) 50 56.456 ± 2.422 100 64.260 ± 0.871200 93.376 ± 0.921 250 96.853 ± 0.982 Gingered Magnolia bark 25 14.922 ±5.040 27.80 extract (cultured for 90 50 94.135 ± 3.192 days) ParchedPuerariae radix 25 25.155 ± 1.542 42.50 extract (cultured for 90 50643.712 ± 3.069  days) 100 100 200 100 250 100 Glycyrrhizae radixextract 25  49.37 ± 1.802 28.40 (cultured for 90 days) 50 62.262 ± 14.68100 100 200 100 250 100 Magnolol (cultured for 3.33 36.36 ± 1.61 5.36 30days) 6.66 55.75 ± 2.11 13.32 77.05 ± 1.70 26.64 100 Amino- Cultured27.5  45.78 ± 2.400 34.90 guanidine for 30 days 55  55.43 ± 4.000 110 73.52 ± 1.750 550  96.41 ± 2.200 Cultured 27.5 39.647 ± 3.406 30.80 for90 days 55 65.712 ± 3.242 110 65.714 ± 5.394 55 89.873 ± 2.554

As can be seen in Table 1, the herbal extract mixture of the presentinvention, when cultured for 30 days, showed a very low IC₅₀ of 18.12μg/ml, indicating a very excellent effect on the inhibition ofproduction of advanced glycation endproducts.

Meanwhile, the extract from each of Euphorbiae radix, gingered Magnoliabark, parched Puerariae radix and Glycyrrhizae radix, when cultured for90 days, showed a low IC₅₀, indicating that it has an excellent effecton the inhibition of production of advanced glycation endprodicts.However, when cultured for 30 days, the single herbal extract was not soexcellent in its effect, suggesting that its effect will be shown uponlong-term administration.

Furthermore, the inventive herbal extract mixture showed a much lowerIC₅₀ than that of aminoguanidine, a positive control group, indicatingthat it has a very excellent effect on the inhibition of production ofadvanced glycation products.

Accordingly, it can be found that the single herbal extract according tothe present invention has an inhibitory effect against the production ofadvanced glycation endproducts, and the inventive herbal extract mixturehas a higher effect than that of the single herbal extract on theinhibition of production of advanced glycation endproducts.

Test Example 2 Effect of Inventive Herbal Extract Mixture on Treatmentof Diabetic Complications

In order to examine the effect of the inventive herbal extract mixtureon the treatment of diabetic complications, the following tests wereperformed.

1. Test Animals

Four-week-old SD male rats weighing 120-140 g were housed in cages atone animal for each cage and allowed for access to general solid foodand water while adapting the animals to an animal room of this institute(the applicant).

The animal room was maintained under the following conditions:temperature 23±3° C.; relative humidity: 50±10%; illumination time: 12hours (a.m. 8 to p.m. 8); ventilation number: 10-20 times/hr; andillumination intensity: 150-300 Lux. During the test period, thetemperature and humidity of the animal room were automaticallycontrolled by a constant temperature and humidity chamber, andenvironmental conditions such as illumination intensity were regularlymeasured. Also, there was no change influencing the tests.

The test animals were divided into the following four groups: (1) anormal group administered with carboxymethyl cellulose (NC+CMC); (2) adiabetes-induced group administered with carboxymethylcellulose(DC+CMC); (3) a diabetes-induced group administered with the herbalextract mixture (DC+HMP); (4) and a diabetes-induced group administeredwith a positive control (Eparlestat) (DC+S11). The diabetes-inducedgroups each consisted of 9-10 animals, and the normal group consisted of5-6 animals. The body weights of the animals were similar between thetest groups.

2. Induction of Diabetic Complications by Streptozotocin

Streptozotocin (STZ) (N-[methylnitrosocarbamyl]-D-glucosamine), which isa chemical substance of breaking selectively β-cells to induce highblood glucose conditions by insulin deficiency, was used to inducediabetes. Particularly, single high dose streptozotocin (SHDS) can beseen to be suitable for a diabetic complication model since itirreversibly induces high blood glucose conditions by the mass necrosisof β-cells.

The test animals were adapted to the animal room and then fastedovernight. Streptozotocin was dissolved in 0.1 M citrate buffer (pH4.5), and just then, injected into the abdominal cavities of the fastedanimals at a dose of 60 mg/kg. At 2 days after administering thestreptozotocin solution, tail vein blood was sampled from the animalsand examined for blood glucose level. Rats having a blood glucose levelof more than 300 mg/ml were determined to be diabetes-induced rats.

As a positive control group, Epalrestat (ON0-2235; referred to as “S11”)which is currently used as an agent for treating diabetic complicationswas used.

(3) General Observation (Changes in Body Weight, and Food and WaterIntakes)

To examine a therapeutic effect against chronic diabetic complications,the rats was continued to maintain in diabetic conditions for 30 days.From day 31, the rats were administered with the drugs for 8 weeks.

Since the herbal extract mixture (HMP) and Epalrestat (S11) used in thetests are insoluble in water, they were dissolved in 1%carboxymethylcellulose before use. The normal group was administeredwith carboxylmethylcellulose so as to eliminate an effect caused bycarboxylmethylcellulose.

For the two diabetes-induced groups other than the normal group, each of1 g/kg of the herbal extract mixture (HMP) and 25 mg/kg of Epalrestat(S11) was dissolved in 1% CMC and administered orally to the rats by asonde daily for 8 weeks.

After administering the samples during the administration period, thetest animals were fasted for at least 15 hours at one day beforeautopsy, and then measured for body weight.

The body weight was measured every day, the feed intake was measured onetime a week, and the water intake was measured every day.

The measurement results are shown in Table 2 below. TABLE 2 Increase inEarly stage (g) End stage (g) body weight (g) NC + CMC 209.26 ± 28.94462.09 ± 32.91 252.83 DC + CMC 154.33 ± 27.26 220.50 ± 64.23 66.17 DC +HMP 153.50 ± 45.06 251.71 ± 40.01 98.21 DC + S11 154.03 ± 45.56 283.24 ±42.60 129.21Note:NC + CMC: normal group + carboxymethylcelluloseDC + CMC: diabetes-induced group + carboxymethylcelluloseDC + HMP: diabetes-induced group + herbal extract mixtureDC + S11: diabetes-induced group + positive control (Epalrestat)

As can be seen in Table 2, the body weight was increased by about 252.83g for the normal group, and only 66.17 g for the diabetes-induced groupadministered with carboxymethylcellulose. Also, the body weight wasincreased by 98.21 g for the diabetes-induced group administered withthe herbal extract mixture (HMP), and 129.21 g for the diabetes-inducedgroup administered with Epalrestat (S11), a positive control. As such,an increase in the body weight of the group administered with theinventive herbal extract mixture was lower than that of the Epalrestat(S11)-administered group, but higher than that of the diabetes-inducedgroup administered with carboxymethylcellulose.

Furthermore, in spite of low body weight, the diabetes-induced groupadministered with carboxylmethylcellulose consumed 2 times more feedthan that of the normal group, and the positive control Epalrestat(S11)-administered group consumed more feed than that of thediabetes-induced group. However, the group administered with theinventive herbal extract mixture consumed slightly less feed than thatof the diabetes-induced group, and the diabetes-induced group ingested 7times more water than that of the normal group.

Also, the group administered with the herbal extract mixture ingestedabout 5 times less water than that of the diabetes-induced group, andthe group administered with the positive control Epalrestat (S11)ingested more water than that of the diabetes-induced group.

(4) Measurement of Organ Weight

One day before autopsy, the test animals were fasted for at least 15hours and then measured for body weight. Then, the animals wereanesthetized with ethyl ether, from which blood was collected via theabdominal aorta. Some of the collected blood was treated with heparin,and some of whole blood was centrifuged at 3,000 rpm and 4° C. for 15minutes. From each of the blood samples, plasma was isolated and storedat −80° C. before use in data analysis.

The kidneys of the animals were removed after washing by perfusion andmeasured for weight. Next, the kidneys were separated into cortex andmedulla, which were rapidly cooled and stored at −80° C. In addition,the liver, lungs, pancreas, spleen and heart were separated and measuredfor weight.

The measurement results are shown in Table 3 below. TABLE 3 Heart LiverSpleen Lungs Kidneys Pancreas NC + CMC Absolute 1.64 ± 0.07 10.60 ± 0.720.79 ± 0.10 1.84 ± 0.23 2.65 ± 0.18 1.13 ± 0.21 weight (g) Relative 0.25± 0.02  2.30 ± 0.12 0.17 ± 0.02 0.40 ± 0.04 0.58 ± 0.04 0.25 ± 0.05weight (%) DC + CMC Absolute 0.84 ± 0.18*** 10.30 ± 1.99 0.46 ± 0.15***1.44 ± 0.24** 2.92 ± 0.04 0.79 ± 0.24** weight (g) Relative 0.37 ±0.06***  4.62 ± 0.71*** 0.20 ± 0.03* 0.65 ± 0.14*** 1.35 ± 0.37*** 0.35± 0.09** weight (%) DC + HMP Absolute 0.88 ± 0.14** 10.71 ± 1.21 0.48 ±0.10*** 1.58 ± 0.21* 2.95 ± 0.48 0.93 ± 0.14 weight (g) Relative 0.35 ±0.03***  4.31 ± 0.48*** 0.19 ± 0.22 0.64 ± 0.08*** 1.18 ± 0.10*** 0.38 ±0.07*** weight (%) DC + H11 Absolute 0.95 ± 0.11 11.63 ± 1.08 0.54 ±0.09 1.55 ± 0.13 3.18 ± 0.30 0.86 ± 0.11 weight (g) Relative 0.34 ±0.03***  4.15 ± 0.40*** 0.19 ± 0.02 0.55 ± 0.06*** 1.14 ± 0.11*** 0.31 ±0.05* weight (%)Note:NC + CMC: normal group + carboxymethylcelluloseDc + CMC: diabetes-induced group + carboxymethylcelluloseDC + HMP: diabetes-induced group + herbal extract mixtureDC + S11: diabetes-induced group + positive control (Epalrestat)Statistical comparison with normal group: *p < 0.05, p < 0.01, and ***p< 0.001.

As can be seen in Table 3, the diabetes-induced group showed an increasein the relative weight of all organs excluding the pancreas, as comparedto the normal group, and particularly, it showed more than two timesincrease in the relative weights of the kidneys and liver. The groupadministered with the inventive herbal extract mixture showed a decreasein the weight of each organ as compared to that of the diabetes-inducedgroup, but this decrease was not significant. Also, the herbal extractmixture showed the similar effect to that of the positive controlEpalrestat (S11).

5. Analysis of Biochemical Factors in Serum

(1) Blood Glucose Lowering Effect

At two-week intervals up to before autopsy, blood was taken from theorbital veins and measured for plasma glucose level with a glucose kit.The plasma glucose level was measured at 500 nm UV by a glucose oxidasemethod.

(2) Improvement Effect on Kidney Function

(a) Blood Urea Nitrogen (BUN)

BUN was determined by measuring absorbance at 580 nm by aurease-indophenol method.

(b) Triglyceride

Triglyceride content was quantified by measuring absorbance at 550 nm byan enzymatic method (POD).

(c) Total Cholesterol Level

Total cholesterol level was determined by measuring absorbance at 500 nmby an enzymatic method.

(d) Creatinine

Creatinine was determined by measuring absorbance at 515 nm by amodified Jaffe's method.

(e) Protein

Protein in serum was determined by BCA assay. Serum was reacted with abicinchonicic acid solution of bicinchonicic acid, Na₂CO₃, NaHCO₃ andC₄H₄O₆Na₂.2H₂O in 0.1N NaOH and a 4% CuSO₄.5H₂O solution and thenmeasured for absorbance at 562 nm.

The measurement results are given in Table 4 below. TABLE 4 TriglycerideTotal cholesterol Glucose level (mg/dl) BUN (mg/dl) (mg/dl) (mg/dl)Creatinin (mg/dl) Protein (μg/μl) NC + CMC 124.93 ± 16.25 15.12 ± 0.5433.38 ± 6.62 63.73 ± 11.05 1.54 ± 0.08 12.63 ± 0.28 DC + CMC 408.79 ±63.10*** 33.13 ± 5.13*** 70.20 ± 13.14*** 54.76 ± 11.85 1.75 ± 0.17*12.47 ± 0.42 DC + HMP 271.22 ± 72.28***## 26.62 ± 3.51***# 50.35 ±16.63# 53.56 ± 6.18 1.57 ± 0.28* 12.35 ± 1.61* DC + S11 197.94 ±76.66*### 23.60 ± 5.15***## 46.18 ± 8.26*## 59.50 ± 13.88 1.22 ±0.16***## 10.79 ± 0.85***#Note:NC + CMC: normal group + carboxymethylcelluloseDC + CMC: diabetes-induced group + carboxymethylcelluloseDC + HMP: diabetes-induced group + herbal extract mixtureDC + S11: diabetes-induced group + positive control (Epalrestat)Statistical comparison with normal group: *p < 0.05, **p < 0.01, and***p < 0.001Statistical comparison with diabetes-induced group: #p < 0.05, ##p <0.01, and ###p < 0.001.

As can be seen in Table 4, the blood glucose levels were 408.79±63.10mg/dl for the diabetes-induced group, and 271.22±72.28 mg/dl for theinventive herbal extract mixture-administered group, indicating that theinventive herbal extract mixture is excellent in the bloodglucose-lowering effect as compared to the diabetes-induced group(p<0.01). The group administered with the positive control Epalrestatshowed a blood glucose level of 197.94±7.666 mg/dl.

Furthermore, the BUN level, an index of the kidney function, was about2.4 times higher in the diabetes-induced group than in the normal group.The BUN level was significantly lower for the inventive herbal extractmixture-administered group than for the diabetes-induced group (p<0.05).

Moreover, the triglyceride level was much higher in the diabetes-inducedgroup (70.20±13.14 mg/ml) than in the normal group (33.38±6.62 mg/dl)(p<0.001), and significantly lower in the inventive herbal extractmixture-administered group (50.35±16.63 mg/dl) than in thediabetes-induced group (p<0.05).

Also, the creatinine level was remarkably increased from 1.54±0.08 mg/dlto 1.69±0.24 mg/dl for the diabetes-induced group, and lowered to1.51±0.26 mg/dl for the inventive herbal extract mixture-administeredgroup. Although the creatinine level is also used as an index of thekidney function, it is not a sensitive measure. Accordingly, even whenglomerular filtration rate is reduced by more than 50%, the creatininelevel remains in the normal range. In view of this characteristic, acreatinine level of 1.51±0.26 mg/dl for the inventive herbal extractmixture-administered group indicates a significant improvement in thekidney function. As shown in Table 3 above, the administration of theinventive herbal extract mixture resulted in a reduction in the kidneyhyprotrophy as compared to the diabetes-induced group, indicating animprovement in the kidney function.

Accordingly, it can be found that the inventive herbal extractmixture-administered group showed not only a reduction in the kidneyhypertrophy but also remarkable reductions in the BUN, triglyceride andcreatinine levels, as compared to the diabetes-induced group, suggestinga significant improvement in the kidney function.

(6) Anti-Cataract Effect

From the eyeballs removed under a microscope, the eye lenses wereseparated and transferred onto 24-well plates each containing 2 ml of asaline solution. Then, the eye lenses were photographed with a digitalcamera. The opacity of the eye lenses was analyzed by the use of animaging system program.

The eye lenses were photographed with a camera and measured for weight.Then, the eye lenses were placed in phosphate buffer (pH 6.9) andhomogenized at 4° C. For use in the measurement of enzymatic activity,some of the homogenized solution was centrifuged at 3,000 rpm for 20minutes, and the supernatant was collected and stored at −80° C. For usein the measurement of content of sorbitol, etc., the remaininghomogenized solution was added with ZnSO₄ and NaOH so as to removeprotein, and centrifuged at 3,000 rpm for 20 minutes, and thesupernatant was collected and stored at −80° C.

The results of visual observation for the eyeballs are shown in FIG. 9,and the results of camera observation for the eye lenses are shown inFIG. 10. Also, the opacity of the eye lenses is shown in Table 5 below.TABLE 5 Eye lens's opacity (20 pixels/m²) Eye lens's opacity (%) NC 39.91 ± 23.36  8.31 ± 5.21 DC 200.53 ± 47.88 66.84 ± 14.89*** DC + HMP136.54 ± 73.52 37.20 ± 20.25**## DC + S11 134.04 ± 45.00 37.38 ±13.56***###Note:NC: normal groupDC: diabetes-induced groupDC + HMP: diabetes-induced group + herbal extract mixtureDC + S11: diabetes-induced group + positive control (Epalrestat)Statistical comparison with normal group: *p < 0.05, **p < 0.01, and***p < 0.001Statistical comparison with diabetes-induced group: #p < 0.05, ##p <0.01, and ###p < 0.001.

As shown in FIG. 9 and Table 10, the diabetes-induced group started toshow cataract from 6 weeks after inducing diabetes. Regarding theeyeball conditions of all the groups on the day of autopsy, the eyeballsof three of seven animals in the diabetes-induced group were covered inwhite, and one animal showed very weak symptoms. In the case of thegroup administered with the positive control Epalrestat (S11), botheyeballs of four of seven animals were covered in while, and one animalshowed weak symptoms in the right eyeball. The reason why as many asfive animals in the positive control Epalrestat (S11)-administered groupshowed cataract symptoms is believed to be because rats subjected toStreptozocin induction two times were as many as two animals. It isgenerally known that when Streptozocin induction is performed two times,an increase in body weight will be low and the eye lens opacity becomessevere. Also, in the case of the inventive herbal extractmixture-administered group, three of seven animals showed cataractsymptoms in both eyeballs.

As can be seen in Table 5, the mean opacity of the eye lenses was8.31±5.21% for the normal group, and 66.84±14.89%, indicating that theeye lens opacity was severe. Also, the positive control Epalrestat (S11)and the inventive herbal extract mixture were administered to thediabetes-induced groups, respectively, they significantly reduced thecataract symptoms to 37.38±13.56% (p<0.001) and 37.20±20.25% (p<0.01),respectively.

Also, as shown in FIG. 11 wherein reference numerals 1, 2, 3 . . .denotes the number of rats, and L or R denotes the right or left eyelens of rats, the eye lenses of the normal group showed an opacity ofabout 20% and were also clear in appearance. An opacity of about 20% canbe seen as normal. The animals of the diabetes-induced group all showedan eye lens opacity of more than 40% and mostly showed an eye lensopacity of more than 60%. Also, rats showing an eye lens opacity of morethan 80% were 3 in number. However, the animals of the inventive herbalextract mixture-administered group mostly showed an eye lens opacity ofabout 40%. Among these rats, rats showing a normal level of the eye lensopacity also existed and rats showing an eye lens opacity of more than60% were 2 in number.

Accordingly, it can be found that the inventive herbal extract mixturewill be effectively used for the prevention and treatment cataractsymptoms caused by chronic diabetes.

Test Example 3 Test of Acute Toxicity in Oral Administration to Rats

To examine the acute toxicity of the inventive herbal extract mixture,the following tests were performed.

Acute toxicity test was conducted on six-week-old specific pathogen-free(SPEF) SD rats as test animals. The herbal extract mixture prepared inExample above was suspended in water and administered orally to eachtest animal group consisting of 2 animals, at a dose of 6 g/kg/15 ml onetime. After administering the test substance, the animals were observedfor death, clinical symptoms and body weight change, and subjected tohematological examination and hematobiochemical examination. Afterautopsy, the animals were visually observed for abnormalities inabdominal and thoracic organs.

As a result, there were no special clinical symptoms in all the animalsadministered with the test substance. Also, there was no dead animal,and in view of the results of the body weight measurement, hematologicalexamination, hematobiochemical examination, etc., a change in toxicitywas not observed.

The above results demonstrated that the inventive herbal extract mixtureis a safe substance which does not show a change in toxicity up to adose of 6 g/kg and has a minimum lethal dose (LD₅₀) for oraladministration of at least 6 g/kg.

Formulation 1: Preparation of Tablets

100.0 mg of each of the medicinal herbal extract prepared in Examples1-3, 90.0 mg of corn starch, 175.0 mg of lactose, 15.0 mg ofL-hydroxypropylcellulose, 905.0 mg of polyvinylpyrrolidone and asuitable amount of ethanol were uniformly mixed and granulized by a wetgranulation method. The granules were added with 1.8 mg of magnesiumstearate and tableted in such a manner that one tablet weighed 400 mg.

Formulation 2: Preparation of Capsules

100.0 mg of each of the medicinal herbal extracts prepared in Examples1-3, 80.0 mg of corn starch, 175.0 mg of lactose and 1.8 mg of magnesiumstearate were uniformly mixed, and filled in capsules at an amount of360 mg for each capsule.

Formulation 3: Preparation of Functional Drinks

Drinks were prepared with the following composition by a conventionalmethod: Horney 522 mg Thioctic acid amide 5 mg Nicotinic acid amide 10mg Sodium riboflavin hydrochloride 3 mg Pyridoxine hydrochloride 2 mgInositol 30 mg Orotic acid 50 mg Herbal extract of the invention 500 mgWater 200 mg.

INDUSTRIAL APPLICABILITY

As described above, the inventive single herbal extract, mixed herbalextract or magnolol inhibits the production of advanced glycationendproducts causing diabetic complications, at a much lowerconcentration than that of aminoguanidine, a positive control group.Particularly, the herbal extract mixture is excellent in a bloodglucose-lowering effect, prevents a reduction in body weight, andinduces not only a reduction in kidney hypertrophy but also significantreductions in BUN, triglyceride and creatinine levels, etc., thusinhibiting the deterioration of kidney functions. Also, the herbalextract mixture remarkably reduces the opacity of the eye lens.Accordingly, the herbal extract mixture can be applied in pharmaceuticalcompositions and functional foods for the prevention and treatment ofdiabetic complications caused by the production of advanced glycationendproducts, including diabetic retinopathy, diabetic cataract, diabeticnephropathy and diabetic neuropathy.

Also, the inventive herbal extract reduces the incidence of oxidativestress as a result of the inhibition of production of advanced glycationendproducts. Thus, the inventive herbal extract can be applied inpharmaceutical compositions and functional foods for the prevention anddelay of aging caused by oxidative stress.

1. A composition for the prevention and treatment of diabeticcomplications, which contains an extract obtained by: crushing anddrying anyone selected from Euphorbiae radix, gingered Magnolia bark,parched Puerariae radix and Glycyrrhizae radix; extracting the driedherbal material with alcohol or aqueous alcohol solution; filtering theextract; and concentrating the filtrate under reduced pressure.
 2. Acomposition for the prevention and treatment of diabetic complications,which contains a mixture of extracts from Euphorbiae radix, gingeredMagnolia bark, parched Puerariae radix and Glycyrrhizae radix, theamount of each of the herbal extracts being 5-85% by weight based on100% by weight of the herbal extract mixture.
 3. A composition for theprevention and treatment of diabetic complications, which contains anherbal extract mixture obtained by: crushing and drying each ofEuphorbiae Radix, gingered Magnolia bark, parched Puerariae radix andGlycyrrhizae radix: mixing the crushed herbal materials together, eachof the herbal materials being used at an amount of 5-85% by weight basedon the total weight of the herbal materials taken as 100% by weight:extracting the herbal mixture with alcohol or aqueous alcohol solution;filtering the extract; and concentrating the filtrate under reducedpressure.
 4. A pharmaceutical composition for the prevention ortreatment of diabetic complications, which contains, as an activeingredient, an extract obtained as claimed in claim
 1. 5. A functionalfood for the prevention or treatment of diabetic complications, whichcontains, as an active ingredient, an extract obtained as claimed inclaim
 1. 6. A pharmaceutical composition for the prevention and delay ofaging, which contains, as an active ingredient, an extract obtained asclaimed in claim
 1. 7. A functional food for the prevention and delay ofaging, which contains, as an active ingredient, an extract obtained asclaimed in claim
 1. 8. A composition for the prevention or treatment ofdiabetic complications, which contains, as an active ingredient,magnolol isolated from Magnolia bark, or a pharmaceutically acceptablesalt thereof.
 9. The composition of claim 8, wherein the magnolol isobtained by: extracting Magnolia bark with 80% ethanol aqueous solutionat ambient temperature for 24 hours; systematically separating theextract in the order of normal-hexane, ethyl acetate and normal-butanol;selecting the normal-hexane layer 20 and separating the selected layerinto pre-fractions by normal silica gel chromatography; comparing thepre-fractions with the standard form magnolol on TLC to determine amagnolol-enriched fraction; and separating magnolol from themagnolol-enriched fraction by silica gel column chromatography.
 10. Apharmaceutical composition for the prevention or treatment of diabeticcomplications, which contains, as an active ingredient, magnololobtained as claimed in claim
 8. 11. A functional food for the preventionor treatment of diabetic complications, which contains, as an activeingredient, magnolol obtained as claimed in claim
 8. 12. Apharmaceutical composition for the prevention and 35 delay of aging,which contains, as an active ingredient, magnolol obtained as claimed inclaim
 8. 13. A functional food for the prevention and delay of aging,which contains, as an active ingredient, magnolol obtained as claimed inclaim 8.